Existing imaging technologies are well-suited to study the behavior and functions of a specific protein ensemble in cells. On the other hand, single molecule based analysis of protein function in living cells is notoriously difficult, as spatio-temporal variations in auto-fluorescence compromise image contrast. Since protein function is often revealed within the context of a living cell or organism, it will be essential to develop new classes of optical probe, imaging microscopy and associated analyses that will lead to dramatic improvements in image contrast at the level of few to single molecules. The objective of the proposed research is to improve the detection of few to single protein molecules in cells in culture and in living organisms by a factor of >100. To meet the objective we will develop new classes of synthetic and genetically-encoded optical switch probes and optimize a Snap-tag approach to target optical switch probes to specific proteins in live cells. In particular these probes will be used for single molecule imaging studies of motor proteins within neuronal cells. The ability to modulate the emission from optical switch probes is key to the development of a paradigm-shifting approach for high-contrast fluorescence imaging that we term optical lock-in detection (OLID) imaging microscopy. The improved image contrast from OLID microscopy is achieved through extracting the fluorescence of an optical switch probe, whose intensity is modulated according to a defined perturbation waveform, from a large background noise environment including autofluorescence;lock-in detection and associated image analysis are performed to extract the signal modulation embedded in the background by generating a map of correlation coefficients, or a correlation image, when displayed on a pixel-by-pixel basis. In this proposal we will also describe new approaches to increase the efficiency of OLID-imaging and the design of new optical probes that are suitable for differentiate detection of specific single protein conjugates from their complexes in living cells. This multi-investigator based research proposal has four aims that focus on (a), improving our earlier classes of optical switch for ensemble and single molecule imaging;(b), developing automated analysis for the control of OLID microscopy and real-time image analysis;(c), using OLID imaging microscopy to study the roles of motor proteins myosin V and myosin VI in nerve cells and to establish how mutations in these motors affect the properties of vesicle transport within neuronal cells. Public Health Relevance: The research describes new classes of optical probes and associated imaging techniques that provide impressive improvements in image contrast for few to single molecule imaging of specific proteins in living cells. These technologies will be used within single molecule imaging studies to investigate the roles of myosin motor proteins in axonal transport of specific types of vesicles within neurons derived from normal mice and those from a mouse model of Alzheimers disease.